title: Identification and characterization of another 4-nitrophenol degradation gene cluster, nps, in Rhodococcus sp. strain PN1.
abstract: 4-Nitrophenol (4-NP) is a toxic compound formed in soil by the hydrolysis of organophosphorous pesticides, such as parathion. We previously reported the presence of the 4-NP degradation gene cluster (nphRA1A2) in Rhodococcus sp. strain PN1, which encodes a two-component 4-NP hydroxylase system that oxidizes 4-NP into 4-nitrocatechol. In the current study, another gene cluster (npsC and npsRA2A1B) encoding a similar 4-NP hydroxylase system was cloned from strain PN1. The enzymes from this 4-NP hydroxylase system (NpsA1 and NpsA2) were purified as histidine-tagged (His-) proteins and then characterized. His-NpsA2 showed NADH/FAD oxidoreductase activity, and His-NpsA1 showed 4-NP oxidizing activity in the presence of His-NpsA2. In the 4-NP oxidation using the reconstituted enzyme system (His-NpsA1 and His-NpsA2), hydroquinone (35% of 4-NP disappeared) and hydroxyquinol (59% of 4-NP disappeared) were detected in the presence of ascorbic acid as a reducing reagent, suggesting that, without the reducing reagent, 4-NP was converted into their oxidized forms, 1,4-benzoquinone and 2-hydroxy-1,4-benzoquinone. In addition, in the cell extract of recombinant Escherichia coli expressing npsB, a typical spectral change showing conversion of hydroxyquinol into maleylacetate was observed. These results indicate that this nps gene cluster, in addition to the nph gene cluster, is also involved in 4-NP degradation in strain PN1.
authors.affiliation: Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Munehiro
authors.initials: M
authors.lastname: Nishimura
authors.forename: Dai-ichiro
authors.initials: D
authors.lastname: Kato
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takeo
authors.forename: Seiji
authors.initials: S
authors.lastname: Negoro
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016227:Benzoquinones / Q000378:metabolism; D002396:Catechols / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D006873:Hydroquinones / Q000378:metabolism; D005810:Multigene Family; D009596:Nitrophenols / Q000378:metabolism*; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D016227:Benzoquinones; D002396:Catechols; D006873:Hydroquinones; D009596:Nitrophenols; C013038:hydroxyhydroquinone; C054093:2-hydroxy-1,4-benzoquinone; D010105:Oxygenases; C001833:4-nitrocatechol; C024836:4-nitrophenol